the mesosome and remains enclosed during its subsequent growth across ... the membranes bounding the mesosome fuse, and further wall thickening ... division process, the nuclear material appears to be associated at one end ..... band (20 A) surrounding the cytoplasm. ..... evidence indicates that it is the inner layer of.
JOURNAL OF BACTERIOLOGY, Oct. 1967, p. 1189-1205 Copyright © 1967 American Society for Microbiology
Vol. 94, No. 4 Printed in U.S.A.
Fine Structure of Bacillus megaterium During Synchronous Growth D. J. ELLAR,1 D. G. LUNDGREN, AND R. A. SLEPECKY Department of Bacteriology and Botany, Biological Research Laboratories, Syracuse University, Syracuse, New York 13210
Received for publication 17 July 1967
A fine-structure study of synchronously dividing Bacillus megaterium revealed the sequence of events involved in the division of the cell. First, a mesosome develops as a concentric fold of the plasma membrane at the site of septum formation. The mesosome contains membrane-bound vesicular structures, 300 to 500 A in diameter, plus a large membrane-bound structure, 2,000 A in diameter. These larger vesicles are peculiar to mesosomes in this stage of division and are not observed in the mesosomes involved in spore septum formation. The transverse septum originates within the mesosome and remains enclosed during its subsequent growth across the cell. An intimate association is observed between mesosome vesicles, mesosome membrane, and the growing edge of the transverse septum. Prior to completion of the septum, the membranes bounding the mesosome fuse, and further wall thickening occurs within the structure formed by this fusion. At this time, the septum only equals the parent cell wall in thickness. The doubling in thickness of the septum, which is required for the production of two normal daughter cell walls, occurs during a second phase of wall thickening, which is characterized by the appearance of a constriction at the base of the septum. As the constriction widens, the wall in this region thickens, forming the typical rounded poles of the daughter cells. Capsular synthesis at the poles occurs during this second phase of wall thickening. Throughout the division process, the nuclear material appears to be associated at one end with a mesosome at or near the pole of the cell and at the other end to the mesosome involved in septum formation. This association frequently takes the form of a stalklike extension of the mesosome penetrating into the chromatin fibrils.
Electron microscopy of the division process in gram-positive bacteria has shown that the growth of the cross wall is preceded by the inward proliferation of the plasma membrane (13, 18, 30, 36, 39). Several workers have observed "peripheral bodies" or mesosomes in the vicinity of the developing cross wall (5, 11, 20, 23), and it has been suggested that they function in some manner in the orderly synthesis of cell wall material. Evidence to date, however, is not adequate to determine the relationship between the mesosome, the infolding plasma membrane, and the new cross wall, nor is it possible to determine the precise sequence of changes in fine structure which occur when the cell divides. The use of a synchronously dividing culture of Bacillus megaterium offers a more useful method for studying both the nature and time of appearance of these morphological changes during cell division. 1 Present address: Department of Microbiology, New York University School of Medicine, New York, N.Y. 10016.
MATERIALS AND METHODS
The organism used was B. megaterium ATCC 19213 grown on a defined sucrose salts medium (SS) prepared according to Slepecky and Foster (37). For these studies, the medium was supplemented by the addition of 0.02%o Trypticase (BBL). Culture technique. Details of the culture procedures and the modified filtration method of Maruyama and Yanagita (25) which were used to obtain division synchrony have been previously reported (19). In the present experiments, when the culture reached late log phase (166 Klett units, measured with a no. 54 filter), the cells were collected on filters and aseptically pooled into a sterile 3-liter Fernbach flask and placed on a reciprocating shaker at 30 C. The entire filtration process took less than 5 min and was performed at room temperature.
Electroni microscopy. Samples (10 ml) of the culture
were removed from the Fernbach flask at intervals of 1 hr and were fixed immediately with osmium by the agar block method of Kellenberger, Ryter, and Schaud (24). The procedures for postfixation and embedding were the same as those previously described for B. cereus (32). Sections were cut on an
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ELLAR, LUNDGREN, AND SLEPECKY
MT-2 Porter-Blum microtome, transferred to carboncoated copper grids, and poststained with lead hydroxide (42). Preparations were examined in a Phillips 200 microscope at 80 kv with a 20-,u objective aperture. Micrographs were made on Kodak (3.25 by 4 inches) Projector slide plates (contrast) and enlarged photographically. Final magnification of the micrographs is included in the figure legends. RESULTS
In the growth system, after inoculation, the cells divided synchronously for 4 hr. At this time, sporulation began and was complete by the 10th hr after inoculation. Details of growth kinetics are published elsewhere (19). Cells sectioned during the period of synchronous division were observed to be in various stages of cross wall formation and to possess a varying number of mesosomes. Structure of the mesosome. In B. megaterium, all mesosomes have the same basic structure and originate as an invagination of the plasma membrane which subsequently expands into the cytoplasm. Although they vary in size, all mesosomes appear to contain vesicular structures 300 to 500 A in diameter (Fig. 1-6) which are themselves bounded by a membrane similar in appearance to the plasma membrane. In this study, including some of the serial sections, concentric membrane lamellae were only rarely seen in the mesosomes; it would appear, therefore, that the structures seen are in fact true vesicles. Cell division and the role of the mesosome. At the outset of cell division, a mesosome develops as a concentric fold of the plasma membrane at the site of future septum formation (Fig. 1, 2, 5). In cross section this therefore frequently appears as two separate structures. At this point in development, the membranes at the base of the mesosome are separated by a considerable distance (2,000 A), and the structure consequently appears to be in open connection with the space between the cell wall and the plasma membrane (Fig. 1, 2). As the mesosome enlarges, a second distinct structure is observed within the mesosome in addition to the 300 to 500 A membrane-bound vesicles. This is a much larger membrane-bound vesicle, approximately 2,000 A in diameter, and is filled with granular material (Fig. 1, 2, 5, 6). The chemical composition of this material has not been determined. Except for these two structures,
J. BACTERIOL.
the mesosome contains no additional visible material. The new cross wall begins to appear at the base of the mesosome, projecting into the cytoplasm at right angles to the old cell wall (Fig. 3, 4, 5, 18, 19). At the same time, the space at the base of the mesosome narrows considerably, so that the only connection between the contents of the mesosome and the extracytoplasmic region is through the 30 to 60 A space which separates the plasma membrane from the cell wall (Fig. 4). This stage and the subsequent events leading to the completion of the new cross wall are characterized by an intimate association between mesosome vesicles, mesosome membrane, and the growing edge of the transverse septum. Figure 4 shows this association to be such that the mesosome membrane is folded through 900 to cover the growing edge. At its base, the newly developing transverse septum measures approximately 300 to 400 A, which is the same thickness as that of the parent cell wall. At the growing edge, however, the septum is generally only half this thickness (Fig. 3, 4). Before the transverse septum is complete, the extension of the plasma membrane over the surface of the mesosome fuses, and the final stage of this wall synthesis occurs within the structure formed by this fusion (Fig. 6). The exact point at which this fusion occurs has not been determined, but it probably occurs early in development when the new wall has progressed half the distance to the cell center. An exact determination of the time of fusion requires further careful analysis of serial sections, since what appear to be separate mesosomes are frequently revealed in serial sections to be fused. After fusion, it is interesting to observe that the resulting structure is bipartite, with each half distributed to opposite sides of the developing septum (Fig. 6). The growing region of the transverse septum is half the thickness of the completed wall (Fig. 6), and the region of synthesis is in intimate contact with the vescular contents of the mesosomes. Even after the new transverse septum was continuous across the cell, it remained only 300 to 400 A thick. If two normal daughter cells are to be formed, this new wall must double in thickness at some later stage. Since this organism is surrounded by a thick capsule (Fig. 20, 21), this too must be formed at a later time. An examination
FIG. 1. Section of a cell at 2 hr demoizstratinig the cell wall (cw); plasma membranze (pm); poly-3-hlydroxybutyrate granule (PHB); nuclear material (ni) arranged in the form of axially disposed filaments or fibrils. A layer of densely stained ribosomes surrountds tlhe niucleus anid extentds to the plasma membrane in the form of strands (R). Thie mesosome (M) at the site of septum formation conitainis 300 to 500 A vesicular structures (vs) together with a 2,000 A vesicle (VS). X 61,000. FIG. 2. Serial sectioni of the cell in Fig. I demonstrating tlhe conitiniuity of mesosome (M) structure. The axially disposed nuclear material (n) is now revealed as a conttinuous filamenit. X 60,000.
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